U.S. patent application number 13/240234 was filed with the patent office on 2012-12-13 for anti-npc2 monoclonal antibodies and a method of detecting fatty liver tissue, cancer cells or cancer tissue by using them.
This patent application is currently assigned to NATIONAL YANG MING UNIVERSITY. Invention is credited to Kuan-Hsuan CHEN, Yi-Ming CHEN.
Application Number | 20120315644 13/240234 |
Document ID | / |
Family ID | 47293505 |
Filed Date | 2012-12-13 |
United States Patent
Application |
20120315644 |
Kind Code |
A1 |
CHEN; Yi-Ming ; et
al. |
December 13, 2012 |
ANTI-NPC2 MONOCLONAL ANTIBODIES AND A METHOD OF DETECTING FATTY
LIVER TISSUE, CANCER CELLS OR CANCER TISSUE BY USING THEM
Abstract
The present invention is related to anti-NPC2 monoclonal
antibodies, which against NPC2 or glycosylated-NPC2; and is related
to a method of detecting fatty liver tissues, cancer cells or
cancer tissues by evaluating the expression level of NPC2 or
glycosylated-NPC2 in the cells or tissues.
Inventors: |
CHEN; Yi-Ming; (Taipei,
TW) ; CHEN; Kuan-Hsuan; (Taipei, TW) |
Assignee: |
NATIONAL YANG MING
UNIVERSITY
Taipei
TW
|
Family ID: |
47293505 |
Appl. No.: |
13/240234 |
Filed: |
September 22, 2011 |
Current U.S.
Class: |
435/7.1 ;
436/501; 530/387.9 |
Current CPC
Class: |
G01N 33/57484 20130101;
C07K 2317/30 20130101; G01N 2800/08 20130101; G01N 33/6893
20130101; C07K 16/30 20130101; C07K 2317/34 20130101; C07K 16/18
20130101 |
Class at
Publication: |
435/7.1 ;
530/387.9; 436/501 |
International
Class: |
G01N 33/53 20060101
G01N033/53; C07K 16/18 20060101 C07K016/18 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 10, 2011 |
TW |
100120384 |
Claims
1. An NPC2 monoclonal antibody, wherein the monoclonal antibody
against N-terminal half 1-40 amino acid (SEQ No. 2) of NPC
protein.
2. The NPC2 monoclonal antibody in claim 1, wherein the NPC2
protein includes glycosylated NPC2 protein.
3. The NPC2 monoclonal antibody in claim 1, wherein the monoclonal
antibody against N-terminal half 31-40 amino acid (SEQ No. 3) of
NPC protein.
4. A method of detecting fatty liver tissue in an organism, which
includes the steps of: (a) Providing a tested sample form a tested
organism and a normal sample; (b) Detecting the expression level of
NPC2 protein or glycosylated NPC2 protein in the tested sample and
the normal sample by the monoclonal antibody of claim 1 and
evaluating the expression level, wherein the expression level of
the tested sample is higher than the expression level of normal
sample means the tested organism is suffered from fatty liver.
5. The method of claim 4, wherein the sample includes blood tissue,
liver tissue or the combination thereof.
6. The method of claim 5, wherein the sample is liver cell.
7. The method of claim 4, wherein the detecting method includes
western-blotting, immunoprecipitation and immunohistochemistry.
8. A method of detecting cancer in an organism, which includes the
steps of: (a) Providing a tested sample form a tested organism and
a normal sample; (b) Detecting the expression level of NPC2 protein
or glycosylated-NPC2 protein in the tested sample and the normal
sample by the monoclonal antibody of claim 1 and evaluating the
expression level, wherein the expression level of the tested sample
is higher than the expression level of normal sample means the
tested organism is suffered from cancer.
9. The method of claim 8, wherein the cancer includes breast
cancer, colon cancer, lung cancer and prostate cancer.
10. The method of claim 8, wherein the detecting method includes
western-blotting, immunoprecipitation and immunohistochemistry.
11. A method of detecting cancer in an organism, which includes the
steps of: (a) Providing a tested sample form a tested organism and
a normal sample; (b) Detecting the expression level of NPC2 protein
or glycosylated-NPC2 protein in the tested sample and the normal
sample by the monoclonal antibody of claim 1 and evaluating the
expression level, wherein the expression level of the tested sample
is lower than the expression level of normal sample means the
tested organism is suffered from cancer.
12. The method of claim 11, wherein the cancer includes hepatic
cancer and kidney cancer.
13. The method of claim 11, wherein the detecting method includes
western-blotting, immunoprecipitation and immunohistochemistry.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This Non-provisional application claims priority under 35
U.S.C. .sctn.119(a) on Patent Application No(s). 100120384 filed in
Taiwan, Republic of China Jun. 10, 2011, the entire contents of
which are hereby incorporated by reference.
FIELD OF THE INVENTION
[0002] The present invention is related to anti-NPC2 monoclonal
antibodies, which against NPC2 or glycosylated-NPC2; and is related
to a method of detecting fatty liver tissues, cancer cells or
cancer tissues by evaluating the expression level of NPC2 or
glycosylated-NPC2 in the cells or tissues.
BACKGROUND OF THE INVENTION
[0003] Niemann-Pick Type C (NPC) disease is a lipid storage
disorder that is characterized by progressive hepatomegaly and
neurodegeneration. The lipid stores in liver, kidney, spleen, bone
marrow and brain to cause disease. The cause of NPC is NPC1 or NPC2
gene mutation induces the accumulation of unesterified cholesterol
in the late endosomes/lysosomes.
[0004] NPC2 is a small soluble glycoprotein containing 150 amino
acids first characterized as a major secretory protein in human
epididymis. NPC2 protein would be interacted with cholesterol in
lysosome to maintain the homeostasis of cholesterol in the body.
Beside, in human and mouse, NPC2 is expressed in liver and secreted
into bile (Klein, Amigo et al. 2006). When NPC2 gene mutates,
cholesterol will accumulate in cell (Frolov, Zielinski et al.
2003). In addition, it has been reported that Asn-38 is never
glycosylated, while Asn-58 and Asn-138 can be glycosylated. In
normal condition, some NPC2 proteins are glycosylated only on
Asn-58, and others are glycosylated both on Asn-58 and Asn-138. The
Asn-58 modification is suggested to be necessary for proper NPC2
targeting to lysosomes, while the Asn-138 is non-essential.
Although all glycoforms are able to bind cholesterol, one report
indicated an aberrant glycosylation pattern for NPC2 in NPC 1
deficient mouse liver and suggested that the cholesterol transport
deficiency in NPC1 disease may arise from defects of glycosylated
forms of NPC2 protein (Chikh, Vey et al. 2004).
[0005] In 1975, the method of producing "monoclonal antibody" is
developed. The antibodies are mono-specific antibodies that are the
same because they are made by identical immune cells that are all
clones of a unique parent cell. Given almost any substance, it is
possible to produce monoclonal antibodies that specifically bind to
that substance; they can then serve to detect or purify that
substance. Production of monoclonal antibodies involving
human-mouse hybrid cells. Monoclonal antibodies are typically made
by fusing myeloma cells with the spleen cells from a mouse that has
been immunized with the desired antigen.
[0006] How to produce a huge amount of anti-NPC2 monoclonal
antibodies and detect cancer by these antibodies is a problem to be
solved.
SUMMARY OF THE INVENTION
[0007] As mentioned above, one of the purposes of the present
invention is to provide an NPC2 monoclonal antibody, wherein the
monoclonal antibody against N-terminal half 1-40 a.a (SEQ No. 2) of
NPC protein.
[0008] Preferably, the NPC protein includes glycosylated NPC2
protein.
[0009] Preferably, the monoclonal antibody against N-terminal half
31-40 a.a (SEQ No. 3) of NPC protein.
[0010] Another aspect of the present invention is to provide a
method of detecting fatty liver tissue in an organism, which
includes the steps of: [0011] (a) Providing a tested sample form a
tested organism and a normal sample; [0012] (b) Detecting the
expression level of NPC2 protein or glycosylated NPC2 protein in
the tested sample and the normal sample by the monoclonal antibody
of claim 1 and evaluating the expression level, wherein the
expression level of the tested sample is higher than the expression
level of normal sample means the tested organism is suffered from
fatty liver.
[0013] Preferably, the sample includes blood tissue, liver tissue
or the combination thereof.
[0014] Preferably, the sample is liver cell.
[0015] Another aspect of the present invention is to provide a
method of detecting cancer in an organism, which includes the steps
of: [0016] (a) Providing a tested sample form a tested organism and
a normal sample; [0017] (b) Detecting the expression level of NPC2
protein or glycosylated NPC2 protein in the tested sample and the
normal sample by the monoclonal antibody of claim 1 and evaluating
the expression level, wherein the expression level of the tested
sample is higher than the expression level of normal sample means
the tested organism is suffered from cancer.
[0018] Preferably, the cancer includes breast cancer, colon cancer,
lung cancer and prostate cancer.
[0019] Another aspect of the present invention is to provide a
method of detecting cancer in an organism, which includes the steps
of: [0020] (a) Providing a tested sample form a tested organism and
a normal sample; [0021] (b) Detecting the expression level of NPC2
protein or glycosylated NPC2 protein in the tested sample and the
normal sample by the monoclonal antibody of claim 1 and evaluating
the expression level, wherein the expression level of the tested
sample is lower than the expression level of normal sample means
the tested organism is suffered from cancer.
[0022] Preferably, the cancer includes hepatic cancer or kidney
cancer.
[0023] Preferably, the detecting method includes western-blotting,
immunoprecipitation and immunohistochemistry.
[0024] Preferably, the sequence of NPC2 is:
TABLE-US-00001 (SEQ NO. 1)
MRFLAATFLLLALSTAAQAEPVQFKDCGSVDGVIKEVNVSPCPTQPCQL
SKGQSYSVNVTFTSNIQSKSSKAVVHGILMGVPVPFPIPEPDGCKSGIN
CPIQKDKTYSYLNKLPVKSEYPSIKLVVEWQLQDDKNQSLFCWEIPVQI
VSHL.smallcircle.
BRIEF DESCRIPTION OF THE DRAWINGS
[0025] FIG. 1A illustrates GEX4T-1-NPC2 plasmid of expressing
GST-NPC2 protein.
[0026] FIG. 1B illustrates pET28a-NPC2 plasmid of expressing
His-NPC2 protein.
[0027] FIG. 2 shows western-blotting analysis of different kinds of
monoclonal antibodies against His-NPC2 and GST-NPC2.
[0028] FIG. 3 shows western-blotting analysis of different kinds of
monoclonal antibodies against NPC2 in mouse epididyme.
[0029] FIG. 4A illustrates epitope region mapping of anti-NPC2
monoclonal antibody (full length of pNPC2-HA).
[0030] FIG. 4B illustrates epitope region mapping of anti-NPC2
monoclonal antibody (1-80 a.a).
[0031] FIG. 4C illustrates epitope region mapping of anti-NPC2
monoclonal antibody (81-151 a.a).
[0032] FIG. 4D illustrates epitope region mapping of anti-NPC2
monoclonal antibody (41-105a.a).
[0033] FIG. 5 shows the detecting of epitope region of monoclonal
NPC2 antibody (3-6B).
[0034] FIG. 6 shows the knockdown effect of shNPC2 in sk-hep1 cells
was detected using anti-NPC2 monoclonal antibody.
[0035] FIG. 7 shows the H&E staining of the MCD diet-induced
steatohepatitis liver tissues from both genders of WT mice.
[0036] FIG. 8 shows the serum ALT levels from 0, 1, 2 and 5 weeks
of MCD fed WT mice.
[0037] FIG. 9 shows the expression level of glycosylated-NPC2 is
enhanced in MCD fed WT mice.
[0038] FIG. 10 shows the IHC staining of NPC2 using monoclonal NPC2
antibody (3-6B).
[0039] FIG. 11 shows the western blot of serum sample MCD fed WT
mice using monoclonal NPC2 antibody (3-6B).
[0040] FIG. 12 shows the immunohistochemical staining of 46 pairs
of human fatty liver and 50 pairs of HCC tissues using monoclonal
NPC2 antibody (3-6B).
[0041] FIG. 13 shows the protein level of NPC2 in breast cancer
cell.
[0042] FIG. 14 shows the protein level of NPC2 in colon cancer
cell.
[0043] FIG. 15 shows the protein level of NPC2 in lung cancer
cell.
[0044] FIG. 16 shows the protein level of NPC2 in prostate cancer
cell.
[0045] FIG. 17 shows the protein level of NPC2 in kidney cancer
cell.
[0046] FIG. 18 shows the protein level of NPC2 in hepatic cancer
cell.
DETAILED DESCRIPTION OF THE INVENTION
[0047] The anti-NPC2 monoclonal antibody of the present invention
can be identified by western blotting, immunoprecipitation and
immunohistochemistry. The epitope region of present invention
performs highly sensitivity to NPC2 or glycosylated-NPC2. Wherein
the sequence of NPC2 is:
TABLE-US-00002 (SEQ NO. 1)
MRFLAATFLLLALSTAAQAEPVQFKDCGSVDGVIKEVNVSPCPTQPCQL
SKGQSYSVNVTFTSNIQSKSSKAVVHGILMGVPVPFPIPEPDGCKSGIN
CPIQKDKTYSYLNKLPVKSEYPSIKLVVEWQLQDDKNQSLFCWEIPVQI VSHL.
[0048] One of aspect of the present invention is to provide a
method of detecting fatty liver tissue in an organism, which
includes the steps of: [0049] (a) Providing a tested sample form a
tested organism and a normal sample; [0050] (b) Detecting the
expression level of NPC2 protein or glycosylated NPC2 protein in
the tested sample and the normal sample by the monoclonal antibody
of claim 1 and evaluating the expression level, wherein the
expression level of the tested sample is higher than the expression
level of normal sample means the tested organism is suffered from
fatty liver.
[0051] Wherein the sample can be, but not limited to blood tissue,
liver tissue or the combination thereof.
[0052] Wherein the sample can be, but not limited to liver
cell.
[0053] Another aspect of the present invention is to provide a
method of detecting cancer in an organism, which includes the steps
of: [0054] (a) Providing a tested sample form a tested organism and
a normal sample; [0055] (b) Detecting the expression level of NPC2
protein or glycosylated NPC2 protein in the tested sample and the
normal sample by the monoclonal antibody of claim 1 and evaluating
the expression level, wherein the expression level of the tested
sample is higher than the expression level of normal sample means
the tested organism is suffered from cancer.
[0056] Wherein the cancer can be, but not limited to breast cancer,
colon cancer, lung cancer and prostate cancer.
[0057] Another aspect of the present invention is to provide a
method of detecting cancer in an organism, which includes the steps
of: [0058] (a) Providing a tested sample form a tested organism and
a normal sample; [0059] (b) Detecting the expression level of NPC2
protein or glycosylated NPC2 protein in the tested sample and the
normal sample by the monoclonal antibody of claim 1 and evaluating
the expression level, wherein the expression level of the tested
sample is lower than the expression level of normal sample means
the tested organism is suffered from cancer.
[0060] Wherein the cancer can be, but not limited to hepatic cancer
and kidney cancer.
[0061] Wherein the detecting method can be, but not limited to
western-blotting, immunoprecipitation and immunohistochemistry.
[0062] Wherein the sequence of NPC2 is:
TABLE-US-00003 (SEQ NO. 1)
MRFLAATFLLLALSTAAQAEPVQFKDCGSVDGVIKEVNVSPCPTQPCQL
SKGQSYSVNVTFTSNIQSKSSKAVVHGILMGVPVPFPIPEPDGCKSGIN
CPIQKDKTYSYLNKLPVKSEYPSIKLVVEWQLQDDKNQSLFCWEIPVQI VSHL.
[0063] As mentioned above, the monoclonal antibodies of the present
invention specific recognize NPC2 and glycosylated-NPC2. Besides,
the present invention provides a method of detecting cancer by the
monoclonal antibodies.
EXAMPLES
Example 1
Generate NPC2 Monoclonal Antibodies
[0064] To generate a series of monoclonal antibodies against NPC2,
purified GST-NPC2 or His-NPC2 were mixed with Freund's complete
(for the initial immunization) or incomplete (for the booster
injections) adjuvant (Sigma Co., St. Louis, Mo., USA) and the
resultant mixture was used as an immunogen. His-NPC2 RP was used as
a screened antigen for antibody arose by GST-NPC2 RP, and vice
versa (FIG. 1). Mouse mAbs were produced by hybridoma technique.
The hybridomas were dispensed into six 96-well plates and cultured
in a HAT medium. The culture supernatants were screened using
enzyme immunoassay (EIA) with GST-NPC2 RP and His-NPC2 RP.
Hybridoma cells with high optic density were confirmed with Western
blot assay immediately. Each well of cells with positive results
were subcloned into a 96-well plate with a cell density of 0.5 cell
per well. The resultant single clone with positive results were
inoculated at a dosage of 5.times.10.sup.6 to a BALB/c mouse which
has been primed with 0.5 ml pristine (Sigma-Aldrich) previously.
Monoclonal antibodies are purified from the mouse ascites with
protein-A antibody purification kits (Pro-Chem Inc. Acton, Mass.)
and concentrated using Centricon Plus-80 columns (Millipore). The
isotype of each mAb was determined using a commercial kit
(SouthernBiotech, Birmingham, Ala.). High-tittered mAbs purified
from ascites were diluted with 0.1M NaHCO.sub.3 (pH=8.6) to a
concentration of 100 .mu.g/ml, and added to 6 ml sterile
polystyrene Petri dishes. After coating overnight at 4.degree. C.
in a humidified container, the plates were blocked with the
blocking buffer (0.1 M NaHCO.sub.3 pH=8.6, 5 mg/ml BSA, 0.02% NaN3,
with a sterilized filter, stored at 4.degree. C.) and incubated for
at least 1 hour at 4.degree. C. M13 phages displaying random
heptapeptides at the N-terminus of its minor coat protein (pIII)
were subsequently added (Ph.D.-7.TM. Phage Display Peptide Library,
New England Biolabs Inc.). The phages bound to the plates were
selected and repeatedly screened for 3 times before they were
subjected to DNA sequencing.
[0065] Anti-NPC2 monoclonal antibodies (including 14-8D. 5-5B,
5-4B, 4-12C, 3-6B, 2-7D, I-5B and 1-2G) had been generated and
characterized in table 1.
TABLE-US-00004 TABLE 1 Anti-NPC2 monoclonal antibodies: 14-8D. 5-
5B, 5-4B, 4-12C, 3-6B, 2-7D, 1-5B and 1-2G Immunoglobulin
monoclonal gene Heavy Light antibodies name chain chain Epitope
1-2G NPC2 IgG2a kappic N-terminal 1~40 amino acid 1-5B NPC2 IgG2a
kappic N-terminal 1~40 amino acid 2-7B NPC2 IgG2a kappic N-terminal
1~40 amino acid 3-6B NPC2 IgG2a kappic N-terminal 1~40 amino acid
4-12C NPC2 IgG2a kappic N-terminal 1~40 amino acid 5-4B NPC2 IgG2a
kappic N-terminal 1~40 amino acid 5-5B NPC2 IgG2a kappic N-terminal
1~40 amino acid 14-8D NPC2 IgG2a kappic N-terminal 1~40 amino
acid
Example 2
Confirm the Reactivity of NPC2 Monoclonal Antibodies
[0066] Using mouse epididymic cells (known as expressing NPC2
protein) as sample to test the reactivity of the antibodies of
example 1 by western-blotting. The result shows in FIG. 2. In
addition, these mAbs can specifically recognize mouse epididyme,
which is a NPC2 abundantly express tissue. In FIG. 2, the result
are 14-8D, 5-5B, pre-immune serum (negative control), 5-4B, 4-12C,
3-6B, positive control, 2-7B, 1-5B and 1-2G from left to right.
Besides, the Mw of GST-NPC2 and His-NPC2 are at the position of 43
and 17. All of the monoclonal antibodies showed a good reactivity
against His-NPC2 and GST-NPC2.
Example 3
The Epitope Region of NPC2 Monoclonal Antibody
[0067] In order to map the epitope region of monoclonal NPC2
antibodies, different length of pNPC2-HA including full-length
pNPC2-HA, N-terminal half (1-80 amino acid), 41-105 amino acid and
C-terminal half (81-151 amino acid) had been transfected into 293T
cells for 24 hrs and harvested for western blot analysis.
[0068] As shown in FIG. 4A-4D, the arrows indicate the epitope
region and the No. 1.about.9 represents the monoclonal antibodies
of 14-8D. 5-5B, 5-4B, 4-12C, positive control, 3-6B, 2-7B, 1-5B and
1-2G respectively. All eight NPC2 monoclonal antibodies can
recognize full length and N-terminal half (1-80 aa) of pNPC2-HA.
While, 41-105 aa and C-terminal half (81-151 aa) of NPC2 were not
the NPC2 monoclonal antibodies binding sites.
[0069] Taken together, all of the anti-NPC2 monoclonal antibodies
recognize amino acids 1-40 of NPC2 protein,
MRFLAATFLLLALSTAAQAEPVQFKDCGSVDGVIKEVNVS (SEQ No. 2).
Example 4
The Epitope Region of Anti-NPC2 Monoclonal Antibody 3-6B
[0070] Using Peptide Screening to identify the epitope region of
anti-NPC2 monoclonal antibody (3-6B), synthesized 4 peptides of 10
aa which includes N-terminal half (1-40 aa) of NPC2. Then,
identified the epitope region by enzyme immunoassay (EIA).
[0071] Coated the synthesized peptides on EIA 96 well plate by 10
ug/ml (in 68 mM NaHCO3 and 32 mM NaCO3, PH 9.6) and blocked by 2%
BSA in PBST. Then, using primary antibody 3-6B worked for 1 hr in
37.degree. C. and washed by PBST (PBS+0.05% TWEEN 20) for 4 times.
Next, adding secondary antibody anti-mouse IgG HRP (1:3000) for 1
hr in 37.degree. C. and washed by PBST (PBS+0.05% TWEEN 20) wash
for 4 times. Then adding OPD and 3N HCl to stop reaction and using
ELx808 enzyme immunoassay analyzer by 490 nm wavelength.
[0072] The result showed in table 2, the OD490 of peptide NPC2-3140
by anti-NPC2 monoclonal antibody (3-6B) are 1.716 and 1.607, the
other peptides are lower than 0.1 (Table 2). The average of OD490
showed in FIG. 5.
[0073] Therefore, the epitope region of anti-NPC2 monoclonal
antibody (3-6B) is 31-40aa of N-terminal of NPC2 and the sequences
is: DGVIKEVNVS (SEQ No. 3)
TABLE-US-00005 TABLE 2 Enzyme immunoassay of anti-NPC2 monoclonal
antibody (3-6B) Optical density, NPCR peptides OD)(average) OD1 OD2
NPC2 01-10aa 0.0775 0.084 0.071 NPC2 11-20aa 0.056 0.054 0.058 NPC2
21-30aa 0.057 0.054 0.06 NPC2 31-40aa 1.6615 1.716 1.607
Example 5
The Sensitivity of Anti-NPC2 Monoclonal Antibody 3-6B
[0074] The sensitivity of anti-NPC2 monoclonal antibody (3-6B) was
detected from shNPC2 transfected Sk-hep1 cells which are known as
NPC2 expressing cells. Transfected shNPC2 into Sk-hep1 cell to
knock down the NPC2 and used transfecting vector with and without
shNPC2 gene to perform western-blotting. Besides, using a-tubulin
as control.
[0075] As shown in FIG. 6, the anti-NPC2 monoclonal antibody can
recognize the NPC2 protein in Sk-hep1 cells but can not detect in
shNPC2 transfected cells.
[0076] Therefore, the anti-NPC2 monoclonal antibody (3-6B) performs
high sensitivity to NPC2.
Example 6
The Expression of NPC2 in MCD Diet Induced Fatty Liver Tissues and
Serum
[0077] It has been reported that feeding mice with a methionine and
choline deficient (MCD) diet results in hepatic steatosis and
steatohepatitis which mimic non-alcoholic steatohepatitis in human.
Therefore, we decided to utilize such model to investigate the
roles of NPC2 in steastosis using our monoclonal NPC2 antibody.
[0078] H&E staining of MCD mice liver tissue followed by 1, 2
and 5 weeks of MCD demonstrated in FIG. 7.
[0079] In FIG. 7, the upper and lower rows represent the H&E
staining of male mice and female mice respectively. And from left
to right are H&E staining of liver tissues from normal, 2 weeks
MCD and 5 weeks MCD mice. Wherein the MCD diet begins to induce
steatosis at the second week, inflammatory infiltration at the
fifth week (shown as arrows).
[0080] As expected, the MCD diet begins to induce steatosis at the
second week, steatohepatitis and abnormal serum ALT levels at the
fifth week (FIG. 8). In both male and female mice, as the MCD
duration increase the ALT levels gets higher. The unit of y-axis is
U/L (U=unit, L=liter).
[0081] Next, using 1:2000 diluted anti-NPC2 monoclonal antibody
3-6B to observe glycosylated-NPC protein level of the liver cell
extract in male and female MCD mice by western blot analysis. As
shown in FIG. 9, that glycosylated-NPC2 was significantly enhanced
as the MCD treatment.
[0082] Besides, using 1:200 diluted anti-NPC2 monoclonal antibody
3-6B to observe glycosylated-NPC of 5 weeks MCD mice by IHC
staining. As shown in FIG. 10, IHC staining of mouse normal and
fatty liver tissues showed that NPC2 displayed an enlargement of
punctuate structure in steatosis region than those
steatosis-adjacent tissues.
[0083] In addition, serum levels of NPC2 were enhanced followed by
5 weeks of MCD diet-induced steatohepatitis model (FIG. 11),
suggesting that the increasing of NPC2 in serum may correlate to
the progression of hepatosteatosis in mice.
Example 7
IHC Staining of NPC2 in Human Fatty Liver and HCC Tissues
[0084] The expression of NPC2 in human fatty liver and HCC tissues
were evaluated using immunohistochemical (IHC) staining. The
results showed that among 56 pairs of steatosis (S) and steatosis
adjacent (SA) tissues, 41 (73%) steatosis tissues had higher NPC2
expression, 10 (18%) had equal level and 5 (9%) had lower
expression than their SA tissue counterparts (FIG. 12, upper
panel). The difference of the NPC2 expression between steatosis and
steatosis adjacent tissues is statistically significant (Wilcoxon
Signed Ranks Test, p=0.02).
[0085] We further analyzed the expression levels of NPC2 in 50
pairs of tumor (T) and tumor-adjacent (TA) tissues from HCC
patients using IHC staining. Importantly, 72% (36/50) tumor tissues
had significant lower expression level of NPC2 than the
tumor-adjacent tissues (FIG. 12, lower panel).
[0086] The statistic result shows in Table 3:
TABLE-US-00006 TABLE 3 IHC staining of NPC2 in human fatty liver
and HCC tissues Human fatty liver tissues S < SA S = SA S >
SA N (%) N (%) N (%) p value 5 (9%) 10 (18%) 41 (73%) 0.02* Human
HCC tissues T < TA T = TA T > TA N (%) N (%) N (%) p value 36
(72%) 12 (24%) 2 (4%) 0.02* S = steatosis tissue; SA =
steatosis-adjacent tissue; T = tumor tissue; TA = tumor-adjacent
tissue.
[0087] Taken together, the data implies that the changes of NPC2
expression may reflect the pathogenesis of steatosis and subsequent
HCC development. It is important to note that the most common form
of chronic liver disease is non-alcoholic fatty liver disease,
which encompasses a clinicopathologic spectrum of disease ranging
from isolated hepatic steatosis to NASH, which can progress to
cirrhosis HCC. Until now, the alteration of NPC2 expression pattern
in fatty liver and HCC did not observe in any publications.
Besides, the level of NPC2 in serum can reflect to the process of
steatohepatitis. Therefore, our data indicated that the
applications of our NPC2 monoclonal antibody are helpful for the
clinical diagnosis and research of non-alcoholic fatty liver
disease.
Example 8
The Expression of NPC2 in Multiple Cancer Tissues
[0088] In order to investigate the clinical application of NPC2
monoclonal antibodies, we detected the expression of NPC2 in
various normal and cancer tissues using IHC staining. FIG. 13-18
shows the IHC staining of cancer tissues in 23 breast cancer
patients, 38 colon cancer patients, 44 lung cancer patients, 60
prostate cancer patients, 33 kidney cancer patients and 50 liver
cancer patients respectively and the statistic result shows in
Table 4-9.
[0089] Comparison between these normal tissues and their
corresponding tumor tissues, NPC2 was significantly up-regulated in
breast, colon, lung, and prostate cancers. In contrast, NPC2 was
significantly down-regulated in kidney and liver. While, there were
no difference between normal and cancer tissues of rectum,
pancreas, esophagus, stomach, ovary and uterine cervix. Taken
together, these results indicated that the aberrant expression of
NPC2 is associated with different cancers.
TABLE-US-00007 TABLE 4 The NPC protein level in breast cancer
patients and in normal people. n = 23 T > N T = N T < N P
value n (%) 18 (78%) 4 (17%) 1 (4%) <0.01
TABLE-US-00008 TABLE 5 The NPC protein level in colon cancer
patients and in normal people. n = 38 T > N T = N T < N P
value n (%) 18 (47%) 14 (37%) 6 (16%) 0.045
TABLE-US-00009 TABLE 6 The NPC protein level in lung cancer
patients and in normal people. n = 44 T > N T = N T < N P
value n (%) 36 (82%) 5 (11%) 9 (20%) <0.01
TABLE-US-00010 TABLE 7 The NPC protein level in prostate cancer
patients and in normal people. T (n = 60) N (n = 9) grade - .+-. 1+
2+ 3+ - .+-. 1+ 2+ P value n (%) 1 (5%) 3 (5%) 22 (37%) 25 (42%) 9
(15%) 1 (11%) 0 6 (66%) 2 (22%) 0.05
TABLE-US-00011 TABLE 8 The NPC protein level in kidney cancer
patients and in normal people. n = 33 T > N T = N T < N P
value n (%) 2 (6%) 0 31 (94%) <0.001
TABLE-US-00012 TABLE 9 The NPC protein level in liver cancer
patients and in normal people. n = 50 T > N T = N T < N P
value n (%) 2 (4%) 12 (24%) 36 (72%) 0.02
[0090] As mentioned above, NPC2 protein level can be an index of
cancer development. Therefore, the present invention of anti-NPC2
monoclonal antibody can be used as cancer detection.
Sequence CWU 1
1
31151PRTHomo sapiens 1Met Arg Phe Leu Ala Ala Thr Phe Leu Leu Leu
Ala Leu Ser Thr Ala1 5 10 15Ala Gln Ala Glu Pro Val Gln Phe Lys Asp
Cys Gly Ser Val Asp Gly 20 25 30Val Ile Lys Glu Val Asn Val Ser Pro
Cys Pro Thr Gln Pro Cys Gln 35 40 45Leu Ser Lys Gly Gln Ser Tyr Ser
Val Asn Val Thr Phe Thr Ser Asn 50 55 60Ile Gln Ser Lys Ser Ser Lys
Ala Val Val His Gly Ile Leu Met Gly65 70 75 80Val Pro Val Pro Phe
Pro Ile Pro Glu Pro Asp Gly Cys Lys Ser Gly 85 90 95Ile Asn Cys Pro
Ile Gln Lys Asp Lys Thr Tyr Ser Tyr Leu Asn Lys 100 105 110Leu Pro
Val Lys Ser Glu Tyr Pro Ser Ile Lys Leu Val Val Glu Trp 115 120
125Gln Leu Gln Asp Asp Lys Asn Gln Ser Leu Phe Cys Trp Glu Ile Pro
130 135 140Val Gln Ile Val Ser His Leu145 150240PRTHomo sapiens
2Met Arg Phe Leu Ala Ala Thr Phe Leu Leu Leu Ala Leu Ser Thr Ala1 5
10 15Ala Gln Ala Glu Pro Val Gln Phe Lys Asp Cys Gly Ser Val Asp
Gly 20 25 30Val Ile Lys Glu Val Asn Val Ser 35 40310PRTHomo sapiens
3Asp Gly Val Ile Lys Glu Val Asn Val Ser1 5 10
* * * * *